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US8846624B2 - Modified protein polymers - Google Patents

Modified protein polymers Download PDF

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US8846624B2
US8846624B2 US12/440,670 US44067007A US8846624B2 US 8846624 B2 US8846624 B2 US 8846624B2 US 44067007 A US44067007 A US 44067007A US 8846624 B2 US8846624 B2 US 8846624B2
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elastin
protein
ggt
proteins
ipavg
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US20100048473A1 (en
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Elliot L. Chaikof
Vincent P. Conticello
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Emory University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/36—Materials or treatment for tissue regeneration for embolization or occlusion, e.g. vaso-occlusive compositions or devices

Definitions

  • the invention generally relates to proteins, particularly elastin-mimetic proteins, and methods of producing and using the same, such as in medical devices and/or medical procedures, and other applications.
  • Cardiovascular disease is a growing concern whose importance in the health care field is evidenced by the effort directed at tissue engineering of artificial blood vessels.
  • Current procedures for alleviating cardiovascular disease such as coronary artery disease involves use of a variety of stents, bypass vessels and/or angioplasty.
  • a common problem with these techniques is the high rate of restonosis that requires one or more additional procedures to ensure blood flow through the region remains effective.
  • One method to assist in reducing subsequent adverse outcome or failure of the procedure is to ensure any implanted device be mechanically matched to the surrounding vessel.
  • any implanted material must also be biocompatible to avoid or minimize an unwanted immune response and anti-thrombogenic to minimize unwanted platelet adhesion.
  • Elastin provides initial elasticity to the vessel wall in the lower strain regime, while collagen prevents overextension of the blood vessel. Accordingly, elastin is an important material that provides elasticity to the blood vessel wall and any implantable medical device in the cardiovascular should model elastin's physical characteristics.
  • elastin-mimetic proteins are generally known in the art (see, e.g., U.S. Pub. No. 2004/0171545 published Sep. 2, 2004), there is a need for such proteins having improved mechanical performance that better match the surrounding in vivo environment while being durable and readily and reliably made.
  • the cardiovascular system has a wide range of operating parameters depending on the location within the vascular tree. For example, the stress exerted on a blood vessel wall in the heart or aorta is very different in terms of magnitude and oscillation than those stresses exerted in the venous system.
  • the venous system tends to be of lower and constant pressure whereas upstream in the arterial system the systolic and diastolic pressures provide continuous and significant cyclic strain on the vessel wall.
  • the pressure and time-dependent forces exerted in a neurovascular defect (e.g., aneurysm) region may be quite different than that in other blood vessels.
  • the disclosure herein includes, inter alia, synthetic elastin-mimetic proteins, and various polypeptides useful for incorporation into the synthetic proteins, that are biocompatible and useful for medical applications including as implantable devices.
  • the elastin mimetic proteins can have selectable physical characteristics so that the proteins (and specifically the medical devices/procedures comprising the proteins) may be tailored to better match the physical environment in which the elastin mimetic proteins are to be implanted.
  • Also disclosed are a variety of related methods for making the proteins, selectively tuning one or more physical characteristics of the protein, methods of casting the protein into a film or fiber network useful for making medical devices and/or coatings thereof.
  • the invention is a triblock protein copolymer having hydrophobic end block regions separated by a hydrophilic center block. Further provided are polymers corresponding to the end region and polymers corresponding to the center region. In various aspects of the invention, chemical cross-linking sites are provided for further tuning of the material's physical parameters. In addition, manipulation of the center and end block regions (relative to each other) provides another mechanism for tuning one or more physical parameters. For example, the respective lengths and/or the hydrophobicity/hydrophilicity are increased or decreased to alter a physical parameter.
  • the invention is a triblock protein copolymer A-B-C, where the end blocks A and C are hydrophobic and the central block B is hydrophilic. In an embodiment, the central block provides elasticity to the protein, and the end block provides plasticity to the protein.
  • the invention is recombinant protein polymers that are biocompatible and have improved mechanical stability and deformation responses and related recombinant methods for expressing and making the polymers.
  • the polymers relate to artificial proteins that are capable of physical and/or chemical cross-links to mimic the mechanical properties of elastin, but are capable of long-term functionality when implanted under relatively demanding in vivo applications, for example.
  • the invention is a synthetic protein triblock copolymer comprising first and second end hydrophobic blocks separated by a central hydrophilic block, wherein:
  • the first and second endblocks of any of the proteins provided herein have the same amino acid sequence or have a different amino acid sequence.
  • At least one the first and second endblocks of the protein comprises the sequence (SEQ ID NO:6, which itself is made from a plurality of 5-mers from SEQ ID NOs:4-5):
  • the central block of any of the proteins provided herein comprise the sequence (SEQ ID NO:7, which itself is made from a plurality of 5-mers from SEQ ID NOs:1-3):
  • the protein triblock copolymer comprises the sequence of B10 (SEQ ID NO:9):
  • any of the proteins disclosed herein are further characterized in terms of the relative lengths of the endblocks to the central block.
  • the protein is described as having an end block length parameter corresponding to the total number of amino acids in the first and second end blocks, and a central block length parameter corresponding to the number of amino acids in the central block.
  • a ratio of the end block length parameter to the central block length parameter has a selected value, wherein the ratio has a value that is about 1, greater than 1, greater than 1.5, from about 1:1 to about 10:1, or about 2:1 to about 10:1.
  • any of the proteins are described in terms of the amount of isoleucine, such as a mole fraction of isoleucine of greater than about 18%, between about 18% to about 25%, or about 20%.
  • any of the proteins are hydrated.
  • Such hydration provides the capacity of at least one of the end hydrophobic blocks to form physical crosslinks that provide improved mechanical stability under sustained or repeated mechanical loading such as, for example, the sustained repeated load experienced by the blood vessel wall, a tissue, or an organ in a living system.
  • any of the proteins are described in terms of any one or more of a physical parameter.
  • any of the proteins have an inverse transition temperature, such as a transition temperature that is between about 15° C. and about 27° C., or selected from a range that is between about 19° C. and about 23° C.
  • the invention is a hydrated film or fiber network comprising any of the proteins disclosed herein.
  • the film or fiber network is cast from a solution comprising TFE or water, such as by electrospinning, and the film or fiber network has a cast temperature.
  • the cast temperature may be of any value so long as suitable elastin-mimetic materials having suitable mechanical properties are obtained, such as a cast temperature selected from a range that is between about 2° C. and about 35° C.
  • any of these films or fiber networks is formed into a tissue engineering scaffold capable of supporting cell growth.
  • a useful property of the proteins disclosed herein is their capacity of having a user-selected physical parameter by selection of appropriate amino acids, amino acid sequences and amino acid configurations.
  • the film or fiber network of any of the proteins optionally have a tunable physical parameter, such as a physical parameter that is a: Young's modulus that is greater than 0.3 MPa; ultimate tensile stress greater than 1 MPa; strain at failure selected from a range that is between 100% and 200%; resilience that is greater than 70% over a strain of 30 to 45%; and creep resistance that is less than 10% at an applied stress greater than 0.3 MPa.
  • a physical parameter such as a physical parameter that is a: Young's modulus that is greater than 0.3 MPa; ultimate tensile stress greater than 1 MPa; strain at failure selected from a range that is between 100% and 200%; resilience that is greater than 70% over a strain of 30 to 45%; and creep resistance that is less than 10% at an applied stress greater than 0.3 MPa.
  • any of the materials described herein may be subject to any one or more post-processing techniques known in the art to further effect a change in one or more physical parameters (e.g., post-processing that changes po
  • any of the films or fiber networks is formed into a medical device that may be implanted into the body, such as a vascular graft. Depending on the location of the vascular graft, however, the desired mechanical properties can be very different. Some applications may require resistance to high loads, other low lows, and others a repeated cycling of loads.
  • An embodiment of the present invention provides the ability to tune any one or more of these parameters by varying one or more of end block to central block length, end block hydrophobicity, center block hydrophilicity, and degree of cross-linking.
  • the invention is a medical device comprising any of the proteins provided herein, such as B9, B10, R1, R2 or R4, or a film or fiber network of any of the proteins.
  • medical devices of particular utility include, but are not limited to, an artificial blood vessel; a stent; a graft; a wound dressing an embolic agent; and a drug delivery device.
  • Any of the medical devices may have a protein, film, or fiber network comprising a protein of the present invention that at least partially coats one or more surfaces of the medical device.
  • the protein, film, or fiber network of the medical device retains physical integrity under sustained mechanical load.
  • any of the proteins comprise one or more chemical cross-linking sites flanking each block. “Chemical cross-linking” refers to covalent interactions, van der Waals interactions, dipole-dipole interactions and/or hydrogen bonding interactions within the proteins that provide the capability of effecting a measurable change in one or more physical parameters, and is different from the “physical cross-linking” arising from the physical interaction of hydrophobic and hydrophilic regions which causes conformational changes.
  • the chemical cross-linking site comprises an amino acid that is lysine. Lysine can be suitably processed to mediate chemical cross-linking, such as by gluteraldehyde or a photocross-linkable acrylate functionalized lysine.
  • the invention is nucleic acid sequence that encodes the any one or more of the first endblock, the second endblock (SEQ ID NO:14), the central block (SEQ ID NO:15) and/or any of the proteins disclosed herein.
  • the nucleic acid sequence encodes the protein having the amino acid sequence of B10 (SEQ ID NOs:9-10), or any blocks thereof (DNA cross-referenced as SEQ ID NOs:11-17, 19 or repeating combinations thereof).
  • the invention is a synthetic protein copolymer triblock having a plurality of chemically cross-linkable sites, such as the protein of SEQ ID NO:33 or:
  • the invention is a synthetic protein copolymer triblock comprising end hydrophobic blocks (SEQ ID NO:23 and/or SEQ ID NO:24) separated by a central hydrophilic block, with a plurality of cross-linkable sites (SEQ ID NO:25), for example the protein having the sequence of lysB10 (SEQ ID NO:26 or 71):
  • the invention is an isolated and purified nucleic acid sequence, that encodes for any one or more of the first endblock (SEQ ID NO:23), the second endblock (SEQ ID NO:24), the central block (SEQ ID NO: 25), repeated any number of times as desired, such as from about 10 to 50, or about 28 as exemplified, or the protein lysB10 (SEQ ID NOs:26 or SEQ ID NO: 71), and mixtures of any of the endblocks and central blocks as disclosed herein repeated any number of times to form copolymers having more than 3 blocks.
  • the invention is a synthetic protein copolymer triblock comprising end hydrophobic blocks separated by a central hydrophilic block, said protein comprising the sequence of R4 or SEQ ID NO:34:
  • the invention is an isolated and purified nucleic acid sequence, the sequence encoding for any one or more of the first endblock, the second endblock, the central block and/or the entire R4 protein, such as the nucleic acid sequence of SEQ ID NO:42.
  • the invention is a peptide capable of establishing elastic-like behavior when incorporated into an elastin-mimetic protein, such as a peptide comprising the sequence R1 protein has in the amino acid sequence of SEQ ID NO. 43.
  • a has a value from about 1 to about 5
  • b has a value from about 1 to about 10
  • c has a value from about 5 to about 50
  • d has a value from about 1 to about 5.
  • R1 has the amino acid sequence of SEQ ID NO:44:
  • the invention is a peptide capable of establishing plastic-like behavior when incorporated into an elastin-mimetic protein, such as a peptide comprising the sequence of R2 protein has in the amino acid sequence of SEQ ID NO. 45
  • a has a value from about 1 to about 5
  • b has a value from about 1 to about 10
  • c has a value from about 5 to about 50
  • d has a value from about 1 to about 5.
  • R2 has the amino acid sequence of SEQ ID NO:46:
  • the invention comprises a multi-block elastin mimetic protein having the formula: R2-R1-R2 or (R2-R1) n ; R1 and R2 are as defined above and wherein n is greater than or equal to 2, or is selected from a range that is between 2 and 10
  • R1 comprises the sequence of SEQ ID NO:44 and R2 comprises the sequence of SEQ ID NO:46:
  • the invention is a medical device, cell, tissue, or organ comprising any one or more of the proteins disclosed herein, such as any one or more of B9 (SEQ ID NO:50), B10 (SEQ ID NOs:9, 26, 33), R1 (SEQ ID NO:44), R2 (SEQ ID NO:46), or R4 (SEQ ID NO:34), any combinations thereof, or spun fiber or fiber networks thereof.
  • the protein is one or more of B10, R1, R2, or R4.
  • a medical device is a vascular graft, such as a shunt.
  • the graft or shunt optionally comprises a base scaffold material that is coated and/or impregnated with any one or more of the proteins or films and/or fiber networks thereof.
  • a shunt that is made of ePTFE.
  • the coating is a multi-layer coating.
  • the medical device comprises a woven collagen graft.
  • the invention is an embolic agent, wherein the embolic agent comprises one or more of the proteins of the present invention, such as any one or more of the amino acid sequences in Table 16 alone or in combination with each other, or SEQ ID NOs:9, 10, 26, 33, 34, 44, 46, 47, 48, 50, B9, B10, R1, R2, R4, or a blend thereof.
  • the embolic agent has an inverse transition temperature, said temperature selected from a range that is between about 19° C. and about 23° C. Such an inverse temperature may be used to readily administer the embolic agent in a liquid form, and upon administration, the embolic agent gels or solidifies.
  • the invention is a method of applying an embolic agent to a patient in need of an embolic agent by providing an embolic agent, wherein the embolic agent is any of the proteins disclosed herein, such as B9, B10, R1, R2, R4 or mixtures thereof.
  • the embolic agent is applied to the patient.
  • the embolic agent is applied in a solid or a gel form.
  • the embolic agent is injectable and has an inverse phase transition temperature that is less than the environment in which the agent is applied, so that upon or after application said embolic agent undergoes a phase transition from liquid to a gel or solid form.
  • the patient in need suffers from a cardiovascular defect.
  • a defect is a neurovascular aneurysm.
  • the invention is a method of producing a fiber network having improved mechanical properties from a triblock copolymer of any of the proteins provided herein, or any mixture thereof.
  • the triblock copolymer is provided and thermally annealed.
  • the triblock copolymer is electrospun, as known in the art (see, e.g., U.S. Pat. App. US-2004-0110439 published Jun. 10, 2004 (ref. 29-01) for various methods of making fibers, fiber networks, and fabrics), to form a fiber or fiber network.
  • the fiber is optionally incubated in an aqueous solution at an annealing temperature sufficient to anneal the fiber network and thereby improve the mechanical properties compared to a fiber network that is not thermally annealed.
  • Examples of specific triblock copolymers has an amino acid sequence selected from the group consisting of B10, B9, R1, R2, R1-R2, R4.
  • the method improves a mechanical property that is an elastic modulus, and the elastic modulus increases by at least 30% compared to a nonannealed fabric.
  • the annealing temperature is greater than 50° C.
  • the method of annealing generates a decrease in water swelling ratio, selected from a range that is between 30% and 70%, or about 50%.
  • the method further comprises preconditioning the fiber network by repeated stress-relaxation cycling. In an aspect, the number of repeats is less than 10, such as between the range of about 4 and about 8.
  • the invention is a method of controllably tuning a creep response parameter in an elastin-mimetic protein triblock copolymer.
  • This is useful for tailoring a protein to the environment in which it will operate (e.g., high load, long term versus low loads).
  • a triblock copolymer A-B-C having a central block region B and endblock regions A and C, wherein the central region is hydrophilic and the endblock regions are hydrophobic is provided. Varying at least one of endblock region size, endblock region hydrophobicity, or both, provides the capability of tuning creep response of the triblock copolymer.
  • the sum of the number of amino acid residues of said endblock regions have a length that is at least two times greater than the number of amino acid residues in the length of the central block region.
  • the triblock optionally comprises any one or more of the proteins disclosed herein, such as B9, B10, R1, R2, R4, etc.
  • the invention is a method of making a shunt for insertion into a patient having a cardiovascular defect.
  • An expanded polytetrafluroethylene (ePTFE) graft having a wall and a lumen is provided.
  • the graft is impregnating and/or coated with any one or more proteins disclosed herein.
  • a protein solution is introduced to a surface wall of the graft under positive pressure so that the protein solution is capable of traversing from one surface of the graft wall to the other surface via a plurality of pores in the graft.
  • appropriate protein solutions include, but are not limited to the protein of any of B9, B10, R1, R2, R4, or any mixture thereof.
  • the protein solution and graft are contacted for a contact time sufficient to ensure the protein solution impregnates the wall.
  • the graft is optionally coated on a surface, such as the lumen facing surface, with the protein solution by introducing the protein solution to the graft lumen-facing wall surface; removing excess protein solution from the lumen; incubating the graft for a coating time period; and optionally repeating the coating steps to generate a multi-layer coated shunt.
  • Any proteins disclosed herein may be provided in the solution, such as a protein comprising B10, R1, R2, and/or R4.
  • the invention is an elastin-mimetic protein polymer, and related methods for synthesizing the elastin-mimetic protein polymers disclosed herein, such as by recombinant expression.
  • One class of elastin-mimetic protein analog comprises analogs with elastic-like behavior based on the sequence:
  • FIG. 1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of B10 copolymer. B10 was run on a 5% SDS-PAGE and stained with Coomassie G250 (BioRad). Molecular weight markers were Precision Plus Protein Kaleidoscope (BioRad).
  • FIG. 2 Differential scanning microcalorimetry of B9 and B10. Signals are shifted for clarity.
  • FIG. 3A shows dynamic shear storage (G′), loss modulus (G′′), and tan ⁇ are plotted as a function of temperature ( ⁇ 2%, ⁇ 1 Hz).
  • FIG. 3B shows dynamic shear storage (G′), loss modulus (G′′), and complex viscosity ( ⁇ *) are plotted as a function of frequency ( ⁇ 2%, 37° C.). The figures shows the rheological behavior of B10 in water
  • FIG. 4 Uniaxial stress-strain analysis.
  • the Young's modulus was 87 ⁇ 9 MPa for TFE-23 and 60 ⁇ 8 MPa for water-4 measured from the first linear range, and was 0.71 ⁇ 0.12 MPa for water-23 film measured from the first 10% of deformation.
  • FIG. 5A shows creep of TFE-23 film. From top to bottom, creep was examined as tensile stress was maintained at 1.0 MPa, 0.8 MPa and 0.6 MPa, respectively.
  • FIG. 5B shows creep of water-4 film. From top to bottom, creep was examined as tensile stress was maintained at 0.8 MPa, 0.6 MPa and 0.4 MPa, respectively.
  • FIG. 5C shows creep of water-23 films. From top to bottom, creep was examined as tensile stress was maintained at 60 KPa, 40 KPa and 30 KPa, respectively. Under 60 KPa stress, creep reached the maximum strain that was allowed on the current testing facility within 12 hours.
  • FIG. 5D shows comparison of the creep behaviors of water-4 films derived from B10 and B9. The short-term creep behaviors demonstrated that films derived from B10 are more stable under mechanical loading. The figures shows the creep analysis of B10 films
  • FIG. 6A shows the influence of preconditioning on resilience of water-4 film.
  • a water-4 sample was cyclically stretched to 30% strain, with an off-loading period of 5 minutes between cycles. Plotted are the stress-strain curves from the first ten cycles of stretches, because stress-strain responses were stabilized after the eight cycles of stretch. Similar responses were also observed for TFE-23 and water-23 samples.
  • FIG. 6B shows the dependence of resilience on the number of preconditioning cycles. Samples cast in different conditions are cyclically stretched to 30% strain, with an off-loading period of 5 minutes between cycles. Plotted is resilience after each cycle against the number of the preconditioning cycles.
  • FIG. 7A shows a water-23 sample was cyclically stretched to 30% strain for 21 cycles, with an off-loading period of 5 minutes between cycles. Plotted are the stress-strain curves from the first 10 cycles, because the material response to the external loading is stabilized after 8 cycles of stretch.
  • FIG. 7B shows a water-23 sample was cyclically stretched to 30% strain and then to 12% strain for 20 cycles, with an off-loading period of 5 minutes between cycles.
  • FIG. 7C shows a water-23 sample was cyclically stretched to 50% strain and then to 30% strain for 20 cycles, with an off-loading period of 5 minutes between cycles. The figures shows the influence of preconditioning on the resilience of water-23 films.
  • FIG. 8A shows a water-4 sample was subjected to cyclic stress of increasing magnitudes (shown in inset), and the deformation history was recorded. Reproducibility was examined on three replicate samples, which were preconditioned at 30% strain for 20 cycles with an off-loading period of 5 minutes between cycles and a two hour recovery time.
  • FIG. 8B shows deformation at the end of each loading (filled circles) and off-loading (open circles) period were plotted against the magnitude of cyclic stress. The figures show the deformation behaviors of preconditioned water-4 films under cyclic stress of increasing magnitude
  • FIG. 9 Deformation behavior of preconditioned water-4 films subjected to a step loading protocol.
  • a water-4 sample was subjected to step stress (shown in inset), and strains at the end of each loading step represented by open circles in water-4 films and by crosses in TFE-23 films were plotted against the magnitude of stress. Reproducibility was examined on three replicate samples, which were preconditioned at 30% strain for 20 cycles with an off-loading period of 5 minutes between cycles.
  • FIG. 10A shows stress-relaxation response for films cast in water at 4° C. and 23° C., following deformation to 30% strain at constant rate of 5 mm/min. The rapid stress relaxation took place in the first few hundreds of seconds. At 20 minutes, stress dropped from 2.6 MPa to 1.1 MPa in water-4 film, and from 100 KPa to 35 KPa in water-23 film, respectively.
  • FIG. 10B shows Stress relaxation responses of TFE-23 film, following deformation at a constant rate of 5 mm/min to 10% and 50% strain.
  • FIG. 11A shows electrospinning experimental setup.
  • FIG. 11B shows Electrospun B9 fibers.
  • FIG. 11C shows electrospun B9 network.
  • FIG. 11D shows electrospun B9 conduit.
  • FIG. 12A shows Young's modulus and FIG. 12B shows ultimate tensile strength of thermally annealed B9 fiber networks, tested at 37° C. in PBS.
  • FIG. 12C shows characteristic uniaxial stress-strain curves for electrospun B9 fabrics generated from ring testing of annealed and non-annealed samples.
  • FIG. 12D shows stress relaxation curves for B9 electrospun fiber networks. Annealing temperature is indicated for each curve.
  • FIG. 13A and FIG. 13B are plates showing Cryo-HRSEM micrographs of B9 electrospun fibers hydrated at 37C.
  • FIG. 13C and FIG. 13D are places showing Cryo-HRSEM micrographs of B9 electrospun fibers annealed at 60C.
  • FIG. 14 Platelet deposition on B9 and ePTFE in a baboon ex vivo shunt model.
  • FIG. 15 1800 bp B9 midblock gene concatemer.
  • FIG. 16 Concatemers created via ligation of monomer library.
  • FIG. 17 Plasmid map of commercial expression vector pQE 80L (Qiagen, Inc). Preparation of the plasmid involves the removal of nucleotides between Bam H I and Hin d III restriction sites within the polyclonal region. Contains an N-terminal His-tag.
  • FIG. 18 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of elastin-mimetic triblock copolymer run on 7.5% SDS-PAGE stained with Coomassie G250. Marker lane: Precision Plus Protein Kaleidoscope (Bio-Rad).
  • FIG. 19 Schematic representation of the baboon femoral arteriovenous shunt model. Test samples are interposed within an exteriorized silicone shunt and In 111 -platelet deposition on test surfaces monitored by scintillation camera imaging using a SPECT system.
  • FIG. 20A shows plain ePTFE.
  • FIG. 20B shows after elastin impregnation.
  • FIG. 20C shows after layer-by-layer elastin deposition,
  • FIG. 20D shows after 24-h flow conditioning in PBS at 37° C.
  • the figures shows macroscopic photographs of unstained (left) and Coomassie-stained (right) graft samples
  • FIG. 21A shows plain ePTFE graft
  • FIG. 21B shows plain water-cast elastin-mimetic film
  • FIG. 21C shows ePTFE graft after elastin impregnation
  • FIG. 21D shows after layer-by-layer elastin deposition.
  • the figures shows infrared ATR spectra from 1800 to 1000 cm ⁇ 1 .
  • FIG. 22 shows scanning electron micrographs of ePTFE vascular grafts processed by critical point drying.
  • Scale bar in FIG. 22A scale is 333 ⁇ m;
  • FIG. 22B scale is 40 ⁇ m;
  • FIG. 22C scale is 2.0 ⁇ m;
  • FIG. 22D scale is 333 ⁇ m;
  • FIG. 22E scale is 40 ⁇ m; and
  • FIG. 22F scale is 2.0 ⁇ m.
  • FIG. 24 Resilience of B9 and B10 scaffolds. Samples are cyclically stretched to 30% strain with a rest period of 5 minutes between cycles. Resilience is measured from the first loading loop for non-preconditioned samples and measured from the 10 th loading loop for preconditioned B10 samples. Data indicate that increased hydrophobicity of endblocks decreases resilience of elastin-mimetic scaffold but mechanical preconditioning enhances resilience.
  • FIG. 25 Stress relaxation of B10 scaffolds under different cast conditions. Samples are stretched to 30% strain at a constant rate of 5 mm/min and then held at this constant strain. Rapid stress relaxation occurs in the first few hundreds of seconds. At 20 minutes, stress decreases from 2.6 MPa to 1.1 MPa, 100 kPa to 35 kPa, and 4.0 Mpa to 1.7 MPa in water-4 scaffold, water-23 scaffold and TFE-23 scaffold, respectively. The first stress drop in TFE-23 scaffold prior to 30% deformation is due to the strain-induced damage effect.
  • FIG. 26 Amino Acid Sequence of Protein-Based Block Copolymer B10.
  • FIG. 27 Amino Acid sequence of triblock copolymer B9, constructed from plastic and elastic [X] elastin sequences.
  • FIG. 28 Molecular Assembly of Modified B10 gene. Crosslinking regions inserted between the plastin and elastin domains in addition to flanking the gene.
  • FIG. 29 Synthesis of repetitive polypeptides via multimerization of DNA monomers, adapted from [25].
  • FIG. 30 Genetic assembly of the gene encoding the triblock copolymer R2-R1-R2, adapted from [49].
  • FIG. 31 Diagram detailing sectioning of 15 ⁇ 33 mm elastin fiber patch for immunohistochemical, electron microscopy, and mechanical analysis.
  • Synthetic refers to an isolated artificial protein that is not normally made by an organism.
  • a synthetic protein may be made by an organism or manufactured outside an organism.
  • the protein may be a recombinant protein in that a organism has been genetically engineered to express the protein or a precursor thereof.
  • Triblock refers to a protein having at least three distinct regions, such as a hydrophobic central block that separates end blocks that tend to be more hydrophilic.
  • a triblock amino acid sequence has additional material inserted between one or more of the blocks or at the block ends.
  • a cross-linkable amino acid or modified amino acid that is capable of cross-linking may be inserted between the blocks to facilitate cross-linkage manipulation.
  • Such chemical cross-linking may be in addition to the physical cross-linking that tends to occur naturally with the amphilic triblocks and provides ability to tailor a mechanical property to the end-application to which the protein may be used.
  • “Creep” refers to a mechanical property of a material that is time-dependent.
  • creep relates to the tendency of a material to permanently deform in response to an applied force or stress applied over time, or a time-dependent deformation of the material under stress.
  • “Inverse transition temperature” refers to the property where a material is a liquid at a lower temperature, but changes state to a gel or solid at a higher temperature. The temperature at which such a change of state begins is referred to as the “inverse transition temperature” and is useful for assisting in placement of an embolic agent into a cardiovascular defect as a liquid initially that later changes to a gel or solid, thereby providing therapeutic benefit.
  • Young's modulus is a mechanical property of a material, device or layer which refers to the ratio of stress to strain for a given substance. Young's modulus may be provided by the expression;
  • Physical parameter refers to a property of the protein or material made from the protein and includes mechanical parameters provided herein (e.g., Young's modulus, bending modulus, compressability, ultimate tensile stress, fracture or failure strain, resilience, permeability, swelling ratio, and other parameters and particularly those parameters used in the art to describe biological systems and materials).
  • a “tunable physical parameter” refers to a parameter that can be controllably adjusted by any of the methods disclosed herein or that depends on the structure or sequence of the proteins that make up a film or fiber network.
  • adjusting the properties of the end and/or central blocks permits tuning of a physical parameter that describes the environment or surrounding tissue in which the film or fiber network is to be used or implanted into (e.g., a blood vessel or a portion of the cardiovascular system).
  • a physical parameter that describes the environment or surrounding tissue in which the film or fiber network is to be used or implanted into
  • further tuning is accomplished by any processing or post-processing known in the art thereby providing further control of the mechanical properties of the medical device.
  • Embolic agent refers to a material that is capable of physically impacting blood flow or altering hemodynamics in and around a blood vessel.
  • the embolic agent may be applied to a blood vessel or blood vessel wall, such as a wall rupture or aneurysm, in a liquid form that subsequently gels or solidifies, thereby displacing or preventing further blood flow in a region.
  • the embolic agent may be applied as a gel, semi-solid or solid in a blood vessel or blood vessel wall, such as a wall rupture or aneurysm to provide a therapeutic benefit.
  • Recombinant protein polymers are synthesized and examined under various loading conditions in order to assess the mechanical stability and deformation responses of physically crosslinked, hydrated, protein polymer networks designed as triblock copolymers with central elastomeric and flanking plastic-like blocks. Uniaxial stress-strain properties, creep and stress relaxation behavior, as well as the effect of various mechanical preconditioning protocols on these responses are characterized. An analysis of viscoelastic behavior demonstrates that an increase in endblock size improves network stability and that mechanical preconditioning significantly enhances the resilience of hydrated films. Furthermore, the presence of three distinct phases of deformation behavior is revealed upon subjecting physically crosslinked protein networks to step and cyclic loading protocols in which the magnitude of the imposed stress is incrementally increased over time.
  • Physically crosslinked protein-based materials possess a number of advantages over their chemically crosslinked counterparts, including ease of processing and the ability to avoid the addition or removal of chemical reagents or unreacted intermediates.
  • physical crosslinks formed as a result of hydrophobic aggregation are often deformed or disrupted under external stresses that may be substantially lower than the forces required to disrupt covalent crosslinks. This feature may limit the capacity of physically crosslinked protein-based materials to retain material integrity under sustained mechanical loading that is often an essential requirement for their application in tissue engineering or regenerative medicine or use as a component of an implanted medical device.
  • Concatemers derived from DNA monomers E and P are inserted into the BsmB I site of their original plasmid containing the monomer cassette. Concatemers encoding 31 repeats of the P monomer and 21 repeats of the E monomer are isolated and identified via restriction cleavage with BamH I and HinD III. Double-stranded DNA sequence analysis confirm the integrity of the concatemers within the recombinant plasmids, which were labeled pE and pP, respectively. Restriction cleavage of plasmid pE with Bbs I/Xma I and plasmid pP with BsmB I/Xma I afforded two fragments, which are separated via preparative agarose gel electrophoresis.
  • Enzymatic ligation of pE and pP afforded the recombinant plasmid pPE, which encoded the diblock PE as a single contiguous reading frame within plasmid pZErO-2.
  • the diblock, pPE is used for subsequent construction of the triblock pPEP using the same biosynthetic scheme. Restriction cleavage of plasmid pP with Bbs I/Xma I and plasmid pPE with BsmB I/Xma I afforded two fragments, which are separated via preparative agarose gel electrophoresis.
  • Enzymatic ligation of pP and pPE afforded the recombinant plasmid pPEP, which encoded the triblock PEP as a single contiguous reading frame within plasmid pZErO-2.
  • the triblock concatemer is liberated from pPEP via restriction cleavage with Bbs I and BsmB I and purified via preparative agarose gel electrophoresis. Enzymatic ligation is used to join the concatemer cassette to the Bbs I sites within the modified polylinker C in plasmid pBAD-A. Double stranded DNA sequence analysis confirms the integrity of the concatemer within the recombinant plasmid, which is subsequently transferred to the expression plasmid, pET-24 (d) via restriction cleavage with Nco I and HinD III. Double stranded DNA sequence analysis confirms the integrity of the concatemer within the recombinant plasmid, which is labeled pB10.
  • Plasmid pB10 encodes the triblock copolymer protein B10 as a single contiguous reading frame within plasmid pET-24 (d) and is used to transform the E. coli expression strain BL21(DE3). This affords a protein triblock containing flanking endblock sequences [VPAVG(IPAVG) 4 ][(IPAVG) 5 ] 33 (SEQ ID NO:7) and a midblock sequence (IPGAG)(VPGAG)VPGEG(VPGAG) 2 [(VPGAG) 2 VPGEG(VPGAG) 2 ] 20 (SEQ ID NO:8) ( FIG. 26 , Table 2). Large-scale fermentation (4 L) is performed at 37° C.
  • TB Terrific Broth
  • kanamycin 50 ⁇ g/mL
  • the fermentation cultures are incubated under antibiotic selection for 48 h at 37° C. with agitation at 225 rpm in an orbital shaker.
  • Cells are harvested via centrifugation at 4° C. and 4,000 g for 20 min and the cell pellet resuspended in lysis buffer (150 mL; 100 mM NaCl, 50 mM Tris-HCl, pH 8.0) and stored at ⁇ 80° C.
  • the frozen cells are lysed by three freeze/thaw cycles.
  • Lysozyme (1 mg/mL), protease inhibitor cocktail (5 mL), benzonase (25 units/mL), and MgCl 2 (1 mM) is added to the lysate and the mixture is incubated at 25° C. for 30 min.
  • the cell lysate is incubated for 12 h at 4° C. and is centrifuged at 18,000 g for 30 min at 4° C. to pellet the cell debris.
  • the target protein is purified from the clarified cell lysate by three to five cycles of temperature-induced precipitation (4° C./37° C.) from 5 M NaCl solution. Dialysis and lyophilization afforded protein B10 as a fibrous solid in isolated yields of 250 mg/L of culture.
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • a recombinant protein that contains flanking hydrophobic endblocks of sequence VPAVG[(IPAVG) 4 (VPAVG)] 16 IPAVG (SEQ ID NO:51) separated by a central hydrophilic block [(VPGVG) 2 (VPGEG)(VPGVG) 2 ] 48 (SEQ ID NO:52) is expressed from E. coli and purified, as detailed elsewhere. Amino acid compositional analysis. B9; Calc. (mol.-%): Ala, 8.1; Glx, 2.4; Gly, 31.9; Ile, 6.4; Pro, 20.0; Val, 31.2. Obs.
  • Rheological data are acquired on an Advanced Rheological Expansion System III rheometer (ARES III, TA instrument, NJ) in parallel plate geometry with a plate diameter of 25 mm.
  • the testing protocol for rheological analysis is detailed elsewhere.
  • 100 mg/mL protein solutions are prepared by adding distilled, deionized water to lyophilized protein at 4° C., shaking the solution for 48 h, and then allowing the solution to equilibrate for 72 h.
  • the gap between parallel plates is adjusted between 0.2-0.35 mm and dynamic mechanical experiments were performed in shear deformation mode.
  • An initial strain amplitude ( ⁇ ) sweep is performed at 4° C. and 37° C. at different frequencies to determine the linear viscoelastic range for the protein polymer.
  • the gelation temperature is determined by heating samples from 4° C. to 40° C. at a rate of 1° C. per minute. Following temperature equilibration at 37° C., viscoelastic properties are examined by a strain sweep at a fixed frequency of 1 Hz and a frequency sweep at fixed strain amplitude of 2%. Experiments are repeated on 5 to 6 samples and representative data presented.
  • films are cast from protein solutions.
  • lyophilized proteins were dissolved at a concentration of 100 mg/mL either in 2,2,2-trifluoroethanol (TFE) at 23° C. or in water at 4° C.
  • TFE 2,2,2-trifluoroethanol
  • the protein solution is then poured into Teflon casting molds and solvent evaporation performed either at 23° C. or at 4° C.
  • Test samples are referred to as TFE-23, water-23, or water-4, indicating the casting solvent and evaporation temperature used for film formation.
  • films are hydrated in phosphate buffered saline (PBS) at 37° C., which contains NaN 3 at 0.2 mg/mL to prevent biological contamination.
  • PBS phosphate buffered saline
  • Hydrated film thickness is typically 0.1 mm for TFE-23 and water-5 films and 0.5 mm for water-23 films.
  • Constant engineering stresses are applied for time periods of up to 30 hours.
  • Four to six samples are prepared for stress-relaxation analysis. Each sample is stretched at 5 mm/min to a fixed strain and the evolution of stress over time is examined. Measurement of stress-relaxation is limited to 30 minutes.
  • Preconditioning protocols Five to six samples cast under different conditions are cyclically stretched to 30% strain for 20 cycles with an off-loading period of 5 minutes between cycles. Water-23 films are also stretched to 30% strain for one cycle and then cyclically stretched to 10% strain for 20 cycles; or stretched to 50% strain for one cycle and then cyclically stretched to 30% strain for 20 cycles with an off-loading period of 5 minutes. Resilience is calculated from loading and under the loading curves.
  • the inverse transition temperature is consistent with protein block structure. Differential scanning microcalorimetry of dilute aqueous solutions of B10 (1 mg/mL) confirms the presence of a single endothermic transition at 21° C. consistent with coacervation of the hydrophobic endblocks ( FIG. 2 ).
  • the inverse transition temperature of B10 is 4° C. lower than that observed for B9 due to an increase in the size and hydrophobicity of the endblocks.
  • the B10 endblocks are nearly twice as large as those of the B9 triblock copolymer and contained a larger mole fraction of isoleucine (20 vs. 16 mol %). Reversibility of the phase transition is confirmed upon repeating microcalorimetry measurements after a 12 h equilibration at 4° C. (data not shown).
  • Block structure alters the Young's modulus of elastin-mimetic triblock protein polymers.
  • Load-extension curves at 37° C. of hydrated B10 films cast either from TFE at 23° C. or water at 4° C. reveals plastic-like deformation behavior, such that, stress increases linearly with increasing strain until a yield point is reached between 2-2.5 MPa, after which elongation occurs with the imposition of a relatively low increment in load.
  • hydrated B10 films produced from an aqueous protein solution cast at 23° C. displays rubber-like behavior with homogeneous deformation occurring in response to low stress levels.
  • microphase separation of the endblocks is further promoted due to a coacervation effect.
  • the contribution of the elastomeric midblock to the mechanical response of the material is enhanced with a corresponding rubber-like stress-strain profile.
  • the influence of casting temperature is demonstrated by the behavior of films cast from water at 4° C.
  • films are produced with a lower degree of microphase separated structure and, therefore, display a higher Young's modulus.
  • the presence of substantially larger endblocks and a relatively smaller midblock accentuates the proportion of plastic-like domains in B10 films and, as a consequence, the generation of materials with a higher elastic modulus under all film forming conditions.
  • Creep responses are modulated by protein block structure.
  • Prior studies characterized creep responses of B9 films cast from water at 4° C. or TFE at 23° C. and revealed substantial deformation responses above 0.2 MPa.
  • time-dependent changes in strain in response to stress will be influenced by the density, size, and chemical nature of the physical crosslinks.
  • the creep response behavior is controllably modified.
  • Creep analysis was performed on hydrated B10 films at 37° C. that were initially produced under a variety of film casting conditions ( FIG. 5 ).
  • Water cast films produced at 4° C. demonstrated limited creep ( ⁇ 10%) over a 20 h observation period at stress levels at or below 0.4 MPa, nearly double the load for B9 films produced under comparable conditions.
  • Films cast from an aqueous solution of B10 at 23° C. demonstrated comparable levels of creep, but at stress levels that were one order of magnitude lower.
  • B10 films cast from TFE demonstrated the lowest level of creep with an observed strain of less than 10% when subjected to a stress of 0.8 MPa; an approximately four-fold greater load than that sustained by similarly fabricated B9 films.
  • Preconditioning by an imposed cyclic stress enhances the resilience of protein polymer films.
  • B10 films Upon subjecting B10 films to periods of repetitive cyclic deformation to 30% strain, we observed the accumulation of residual deformation and a decline in peak stress that stabilized after several cycles ( FIG. 6 ).
  • resilience was significantly enhanced over 10 loading cycles with an increase from 11 ⁇ 2% to 30 ⁇ 2% for TFE-23 films, from 18 ⁇ 2% to 39 ⁇ 2% for water-4 samples, and from 35 ⁇ 2% to 51 ⁇ 2% for water-23 films.
  • the greatest increase in resilience largely occurred after the first loading cycle, presumably due to stabilization of load induced changes in microstructure.
  • the first phase which extends up to an imposed load of 1.2 MPa over a 30 h period, is characterized by small elastic deformation responses, as both the total and residual deformations are small.
  • the second phase over a load range between 1.2 and 2.7 MPa, both the total and residual deformation increase linearly with increasing magnitude of cyclic stress and appreciable residual deformation is observed.
  • a more rapid increase in the total and residual deformation occurs in the third phase consistent with disruption of physical crosslinks.
  • Films examined under step loading also display three similar phases of deformation behavior ( FIG. 9 ). Remarkably, strain levels at each transition point are similar for both protocols, although stress levels were significantly different.
  • Three phases of deformation behavior are also observed for preconditioned films cast from TFE at 23° C.
  • the deformation behavior in the first phase may be attributable to an initial stretching of polypeptide bonds.
  • bond stretch is limited and further deformation must arise from conformational changes in the polymer chain, which likely occurs in the second phase of deformation.
  • Differences in the stress required to induce conformational changes of protein polymer within films processed under different casting conditions are likely related to differences in the mixing of semi-rigid endblocks and flexible midblocks that create energy or stereoelectronic barriers.
  • substantial film deformation is observed after an initial 22-25% strain, which appears to designate the stress level associated with disruption or damage to physical crosslinks. Given that samples are preconditioned at 30% strain for 20 cycles, these data suggest that “new” disruption or damage may occur when deformation approaches or exceeds preconditioning strains.
  • Micro-DSC and rheology studies confirm the presence of an inverse-temperature transition for the elastin-mimetic protein polymer B10 in aqueous solutions with gelation of concentrated solutions at ambient temperatures.
  • Mechanical analysis particularly studies of creep behavior, demonstrate enhanced mechanical stability of physically crosslinked protein networks derived from B10 compared to a triblock copolymer designed with a lower relative content of hydrophobic, plastic-like endblocks.
  • resilience is significantly enhanced by mechanical preconditioning.
  • Newly designed tests consisting of cyclic loading of increasing magnitude and step loading further reveal the presence of three phases of deformation behavior, which likely correspond to peptide bond stretching, conformational changes of polypeptide chains, and disruption of physical crosslinks.
  • the breakage of physical crosslinks strongly depends on the imposed pattern of load, as well as preconditioning protocols.
  • CVD cardiovascular disease
  • This work can be divided into four areas: (i) to synthesize a family of recombinant elastin-mimetic proteins; (ii) to define their molecular level structure-property relationships; (iii) to develop nanofabrication strategies to create organized fiber networks, and (iv) to characterize the capacity of these artificial proteins for the generation of non-thrombogenic small diameter blood vessel substitutes with mechanical properties that closely match those of native blood vessels. Utilizing recombinant proteins based on consideration of the structural properties of the native matrix leads to the creation of vascular conduits with better defined mechanical properties and enhanced biodegradation with improved clinical performance characteristics.
  • nanofiber protein networks comprised of recombinant elastin proteins provides a rational approach for generating a tissue engineered vascular graft with enhanced biostability and mechanical properties that closely match those of a native artery.
  • elastin-mimetic protein polymers capable of forming both physical and chemical crosslinks.
  • elastin-mimetic fibers are produced with controlled elastomeric properties and enhanced biostability through appropriate choice of recombinant peptide sequences that facilitate both chemical and physical crosslink formation.
  • Elastin-mimetic fiber networks have sufficient biostability for use in a vascular construct.
  • a recombinant protein fiber patch retains initial elastomeric properties after in vivo implantation.
  • vascular graft design has adapted more of a tissue engineering approach with new graft design inspired by characteristics of the arterial wall.
  • Decellularized allo- and xenogeneic tissue have alternatively been investigated as materials for vascular grafts. These decellularized natural matrices contain the intact extracellular matrix and associated attachment proteins and have been used to produce structures with increase degradation resistance, decreased thromobgenicity, and decreased inflammatory reactions.
  • Human umbilical vein, bovine carotid artery and small intestine submucosa, chemically crosslinked using gluteraldehyde, have been employed in clinical application though their use has been limited due to suboptimal patency rates via dilation and aneurysm formation [19-24].
  • recombinant proteins that mimic structural matrix proteins can be engineered with a precisely tailored design to modulate tensile strength, elastic modulus, viscoelasticity, and in vivo stability, as well as desired host response. These mimics are optimal candidates in the design of the next generation vascular graft.
  • the inherent elasticity of blood vessels arises from the structure of the medial layer.
  • the media is composed of concentric layers of elastic lamellar units each composed of smooth muscle cells, elastin fibers, and collagen fibrils. Elastin and collagen function in a concerted action in response to imposed deformations. Elastin is primarily responsible for distensibility and elastic recovery of the vessel in the low-strain regime while collagen responds by limiting deformation during excessive strain [31-35].
  • the lamellar unit of the aortic media serves as a foundation in the design of a vascular graft prosthetic [36-38].
  • the elastin protein network appears to be integral to mechanically match the native blood vessel and for the prevention of intimal hyperplasia and potential graft failure.
  • Native elastin is a highly insoluable matrix protein that is responsible for providing extensibility and resilience to most tissues of the body.
  • elastin fiber networks appear in large densities (over 50%) and function to provide resilience to the artery to absorb dynamic systolic stresses of the cardiac cycle and to release energy in the form of blood pressure during diastole [39]. Therefore, elastin networks maximize the durability of tissues that are loaded by repetitive forces by minimizing the conversion of mechanical energy to heat which would ultimately result in tissue damage [35].
  • elastin creates an environment which promotes proper cell function. Specifically within the vascular system, elastin regulates smooth muscle cell phenotype and proliferation, and in this way is responsible for stabilizing arterial structure [39-41].
  • Elastin fibers appear to exist as two morphologically different components, a highly isotropic amorphous elastin constituent within an organized microfibrilar scaffold [42]. Understanding the mechanism of fiber assembly in native elastin is limited. Fiber assembly appears to take place in proximity to the cell membrane where microfibrils appear first, grouped in small bundles. Amorphous elastin is synthesized by smooth muscle cells as the soluble monomer, the 72 kDa precursor tropoelastin, and is secreted within each fiber bundle. Here it is organized into insoluble networks reminiscent of natural rubber. Microfibrils function to properly align tropoelastin to facilitate enzymatic crosslinking via oxidation by lysyl oxidase [43].
  • tropoelastin The distinctive composition of tropoelastin affords unique physical properties of this structural protein.
  • Tropoelastin is rich in glycine (33%), proline (10-13%), and other hydrophobic residues (44%) rendering elastin an extremely hydrophobic protein [44].
  • Tropoelastin contains distinct crosslinking and hydrophobic domains.
  • Crosslinking domains are alanine rich, containing pairs of lysine residues facilitating intermolecular crosslinking. Specifically, lysine residues are separated by either two or three alanine residues allowing for retention of an a-helical conformation in this region. The sequence within the crosslinking domains appears to be conserved as a consequence of the conformational constraints of crosslinking [43].
  • the hydrophobic domains within tropoelastin are composed of three-quarters of valine, glycine, proline, and alanine. Investigations have determined that precise sequence and size of this region are not critical for appropriate function. However, the total size of the protein polymer, 750-800 residues, is highly conserved among species [43].
  • this domain is responsible for facilitating fiber formation through coacervation phenomena, behaviors consistent with native elastin.
  • Spectroscopic analysis has revealed that native elastin, and likewise, protein polymers containing this repeat, exhibit ⁇ -turns and helical ⁇ -spiral conformations and display an inverse temperature transition defined by the generation of a more ordered system upon increasing temperature. This loss of entropy is a consequence of protein folding into ⁇ -spiral conformation and the subsequent reorientation of water from the elastin chain [45].
  • VPGXG amino acid in the fourth (X) position
  • glycine and proline residues are preserved the structure and function of elastin is maintained [48].
  • This discovery has led to the generation of recombinant elastin analogs designed for biomedical applications. For instance, Conticello et al have employed recombinant techniques to design amphiphilic elastin protein polymers consisting of hydrophobic and hydrophilic domains. Through precise sequence design and control of processing conditions, these elastin analogs exhibit a wide range of properties advantageous for biomedical applications, as micelles or physically crosslinked hydrogels [49, 50]. Additionally, groups have incorporated cell binding domains, RGD or REDV, into elastin sequences to functionalize elastin matrix components for endothelial cell attachment [51, 52].
  • elastin In its native form, elastin is present as a network of elastic fibers crosslinked through lysine residues. Characteristically, crosslinking of native elastin is accomplished via enzymatic modification of amino acid side chains of lysine residues in the solid state, i.e. after secretion by cells into the extracellular space. Briefly, crosslinks are formed through the deamination of the ⁇ -amino group of the lysine side chains by the enzyme lysyl oxidase.
  • the reaction occurs in two ways: (i) the reactive aldehyde group condenses with a second aldehyde residue to form allysine aldol or (ii) with the ⁇ -amino group on the lysine to form dehydrolysinonoleucine. These two precursors condense to form the pyridium cross-links esmosine and isodesmosine [13].
  • Electrospinning is a technique for generating fibers with diameters ⁇ 1 ⁇ m. Briefly, the electrospinning technique relies on electrostatic forces to produce sub-micron diameter fibers from protein solutions. A high voltage is applied to a spinneret while a protein solution is slowly being pumped through it. This induces evenly dispersed charges in a pendent drop at the tip of the spinneret, relaxing the fluid surface.
  • elastin-mimetic protein polymers can be designed to facilitate both covalent and physical crosslink formation thus enhancing static and dynamic material behavior. These protein polymers may be formulated into nano-fiber networks with improved compliance, resilience, creep, stress relaxation and biostability. Significantly, this strategy can be integrated into schemes which are ultimately driven either by a desire to generate a cell containing arterial construct or a non-thrombogenic acellular conduit.
  • Data are divided into three areas (i) characterization of first generation elastin-mimetic protein polymers reformulated as fiber networks, (ii) genetic modification of first generation elastin-mimetic protein polymers, and (iii) synthesis of second generation elastin-mimetic protein polymers.
  • B9 studies provide understanding of the relationship between macroscale material properties and microscale features, such as block size and sequence, in engineered proteins [69]. More recent work investigated modulating mechanical properties of B9 films by preferential solvent casting and the impact of casting conditions on static and transient properties of B9 films [70]. These investigations serve as the foundation for subsequent B9 studies and also in the rational design of second generation proteins.
  • Lyophillized B9 protein is reformulated into fiber networks using electrospinning techniques ( FIG. 11A ).
  • a solvent system is employed to allow for interphase mixing of the incompatible blocks of the copolymer on the nanoscale which influenced and enhanced B9's material properties as fibers.
  • Sub-micron diameter B9 fibers are produced from a 12 wt % protein solution using a trifluoroethanol (TFE) solvent system ( FIG. 11 B,C).
  • TFE trifluoroethanol
  • Time-Dependent Mechanical Properties of B9 Fiber Networks When deformation is held constant, a relaxation of the imposed tensile stress is observed. This phenomenon is a result of the disappearance of frictional forces, rearrangement of polymer chains, and possibly micro-damage to the protein. Stress relaxation of B9 fiber networks reveals rapid relaxation of imposed tensile stress following deformation to 64% strain. The stress relaxation took place in the first two hundred seconds. At ten minutes, engineering stress had dropped approximately 45% and stabilized indicating structural re-orientation of anisotropic fibers in the direction of deformation followed by conformational rearrangements of protein chains and network entanglements ( FIG. 12D ).
  • Solid-State Circular Dichromy (CD) spectroscopy and Attenuated Total Reflectance Infrared (IR) spectroscopy are used to investigate potential changes in secondary protein structure induced by thermal annealing (data not shown). Data indicates only subtle changes in secondary structure.
  • Cryo-High Resolution SEM is used to inspect the hydrated morphology of electrospun fiber networks.
  • a freeze-drying protocol was designed to remove of water from the surface of the specimen leaving bulk water/ice at larger depths.
  • Cryo-EM sample preparation indicates similar effects of annealing on water content as longer freeze drying times a necessary to remove bulk water and the hydration shell from non-annealed fibers.
  • a comparative analysis of annealed and non-annealed networks reveals subtle differences in microstructure ( FIG. 13 ). Annealing appears to increase the degree of interpenetration of the elastic and plastic blocks, as observed by a loss of molecular architecture in fibers receiving the annealing treatment (FIG. 13 C,D).
  • first generation protein supports the proposed mechanistic basis that physical crosslinks, afforded by the presence of relatively rigid endblock domains, provides a mechanism for tailoring protein polymer mechanical responses.
  • the current mechanical responses of these elastin-mimetic proteins i.e. Young's modulus, tensile strength, creep, and resilience
  • These first generation proteins provide a further basis for the design of new recombinant proteins (“second generation proteins”) that are capable of both covalent and physical crosslinks.
  • second generation proteins new recombinant proteins
  • Table 5 outlines proteins used in subsequent experiments and their classification as a first or second generation protein.
  • Chemically crosslinkable sites are incorporated within the B10 polymer chain at specified locations and gluteraldehyde crosslinked.
  • An adaptor is prepared to incorporate a single lysine near the N-terminus and a pair of lysine residues at the C-terminus of the gene (Table 7; SEQ ID NO:56). This scheme provides four crosslinking sites: three from the lysine side chains and one from the amino termini.
  • the B10 genes are ligated into the adapter sequence within an expression plasmid.
  • an insert containing a pair of lysine residues are designed for incorporation of crosslinks between the elastin and plastin blocks.
  • insert and adaptor sequences are developed and the molecular re-assembly is delineated as described in FIG. 28 .
  • R1 and R2 Proteins designated R1 and R2 are specifically designed to facilitate physical and/or covalent crosslinking. Specifically, lysine (K) residues will be incorporated at selective sites to facilitate chemical (e.g. gluteraldehyde) crosslinking with precise control over crosslink density.
  • Two unique sequences were designed for both R1 and R2 based on preferred codon usage to enable expression from both E coli and Pichia expression systems. Coding sequences for these analogs are outlined in Table 8 (SEQ ID NOs:57, 59, 61, 63).
  • these proteins SEQ ID NOs:58, 60, 62, 64
  • R1, R2 SEQ ID NOs:44, 46
  • any of the proteins disclosed herein may be applied as formulated blends of one another and optional other components as desired.
  • R1 and R2 protein polymers are synthesized using a genetic engineering strategy which affords near absolute control of macromolecular architecture.
  • the plastic-like and elastic-like segments were designed independently following an identical protocol as described herein and detailed in the ‘Methods’ section.
  • DNA monomer units encoding R1 and R2 and concatemerization of this cassette produce a family of genes differing in size by multiples of the repeat unit (75 bp) ( FIG. 16 ). Additionally, cloning of concatamers into the pZero-1 cloning plasmid (Invitrogen) and screening for requisite sizes via double digestion and agarose gel analysis are performed.
  • R1 E coli (BL21) GTA CCT GGT ATT GGC GTT CCG GGT ATC GGT GTG CCA GGC ATC GGT GTA CCG GGT ATT GGC GTT CCA GGC ATT GGC Pichia (XL100) GTT CCA GGT ATT GGT GTC CCA GGA ATC GGT GTT CCT GGA ATT GGA GTC CCA GGT ATT GGA GTT CCA GGT ATA GGT
  • R2 E coli (BL21) ATT CCG GCT GTT GGT ATC CCA GCT GTT GGT ATC CCA GCT GTT GGC ATT CCG GCT GTA GGT ATC CCG GCA GTG GGC Pichia (XL100) ATT CCA GCT GTT GGT ATC CCT GCC GTC GGT ATT CCT GCT GTT GGA ATC CCA GCA GTC GGT ATT CCA GCC GTT GTT GTT GGT ATC CCT GCC GTC GGT ATT CCT
  • elastin-mimetic fibers are produced with controlled elastomeric properties and enhanced biostability through appropriate choice of recombinant peptide sequences that facilitate both covalent and physical crosslink formation.
  • modified B10 is redesigned to include crosslinking sites flanking each block of the gene, such as the sequence that is or comprises SEQ ID NO:26:
  • the second class incorporates both physical and covalent crosslinks. Specifically, protein polymer triblocks are synthesized based on the sequence SEQ ID NO:49:
  • Static mechanical properties are characterized at 37° C. in PBS using model fiber networks, sectioned from electrospun tubes. Stress-strain properties, such as ultimate tensile strength, maximum strain at failure, Young's modulus, as well as mechanical hysteresis, compliance, and % resilience (i.e. the ability of the material to store energy without permanent deformation) is evaluated by uniaxial ring testing.
  • Transient mechanical behavior is defined by stress-relaxation (fixed strain) and creep (fixed stress) studies at small deformations in order to define instantaneous, time-dependent and viscoelastic material behavior. Using a Dynamic Mechanical Thermal Analyzer (DMTA, TA Instruments) these tests are conducted under physiologically relevant conditions.
  • DMTA Dynamic Mechanical Thermal Analyzer
  • Elastin fiber networks that most closely meet target biomechanical endpoints summarized in Table 11 are selected for further biostability studies. Mechanical values comparable to the elastin component of the arterial wall are the desired objective.
  • Fiber network architecture options Experimental parameters deemed fixed (Table 9-fiber diameter, orientation, pore size) are modulated as needed. Oriented electrospun fibers can be generated as needed. If necessary, controlled fiber orientation provides a capability to generate a more robust elastomeric construct.
  • Elastin-mimetic fiber networks have sufficient biostability to be used in a vascular construct.
  • a recombinant protein fiber patch retains initial elastomeric properties after in vivo implantation.
  • IVC inferior vena cava
  • Vascular prosthesis studies Implant studies to assess preclinical performance of small diameter (4 mm i.d., 10 cm length) elastin-collagen composite conduits as both acellular and endothelialized bioprostheses exist.
  • a single-stranded oligonucleotide corresponding to a monomer repeat unit was chemically synthesized (Sigma Genosys, Inc). The lyophilized sequence was resuspended in elution buffer (10 mM tris-HCl, pH 8.5) to a final concentration of 0.5 ug/uL. DNA Polymerase I Klenow fragment was utilized in a primed extension of the oligonucleotide template for the second strand synthesis yielding the double stranded cassette of the monomer repeat unit.
  • LSLB low salt LB
  • agar plates 5 g tryptone, 2.5 g yeast extract, 2.5 g NaCl, 7.5 g agar, 200 mL ddH 2 O, pH 7.5
  • Twenty-four transformants were used to inoculate separate 7 mL LSLB cultures supplemented with Zeocin (50 ug/mL) for antibiotic selection. Cultures were rotary incubated at 37° C. for 12-14 hours.
  • Plasmid DNA was isolated using Qiagen Spin MiniPrep protocol (QIAGEN, Inc). Clones were screened by a Bam H I/Hin d III double digestion. Positive clones were identified by analysis of cleavage products with agarose gel electrophoresis (2% GTG Nuseive, 1 ⁇ TBE buffer) and confirmed by automated DNA sequence analysis (Center for Fundamental and Applied Molecular Evolution, Emory University).
  • Recombinant plasmids containing correct inserts of for each of the selected sequences were re-transformed into competent Top 10F′ cells and plated on LSLB agar plates under Zeocin antibiotic resistance. A single colony from each plate was used to inoculate 500 mL LSLB medium and grown overnight at 37° C. in an orbital shaker at 225 rpm. Preparative amounts of plasmid DNA was isolated using QIAfilter Plasmid Maxi protocol (QIAGEN, Inc). Monomer cassettes were excised from the plasmid via sequential digestion by Bbs I (10 U/uL) and Bsm B I (5 U/uL) restriction enzymes.
  • Fragments of 75 bp were isolated via preparative gel electrophoresis (4% GTG NuSieve, 1 ⁇ TBE buffer), extracted using Aimcon Ultrafree Centrifugal Filter Units (Milipore) and isolated via enthanol precipitation.
  • Multimerization reactions utilized 3.0 ug of the BbsI/Bsm BI digested DNA and ligated monomers end-to-end via T4 DNA ligase. Multimer mixtures were separated by size using agarose gel electrophoresis (1% agarose, 1 ⁇ TBE buffer). Concatemers were excised in blocks, ⁇ 500 bp, 500-1000 bp, 1000-3000 bp and purified using Zymoclean Gel DNA Recovery protocol (Zymo Research, Inc). Multimers of 500-1000 and 1000-3000 bp in size were ligated into the acceptor plasmid at the Bbs I site at 16° C. for 16 hours.
  • the acceptor plasmid was prepared from the pZErO-1 plasmid containing the original monomer repeat unit associated with each gene, digesting with Bbs I, and dephosphorylated via SAP (Shrimp Alkaline Phosphatase) to prevent self ligation.
  • Ligation mixtures were used to transform competent Top 10F′ cells and 100 uL of the transformation mixture was plated on LSLB/Zeocin agar plates. DNA from positive clones were isolated via MacConnell automated miniprep and screened through double digestion using Bam H I and Hin d III restriction enzymes. Clones of predetermined sizes were isolated.
  • the recombinant techniques described above were employed in the generation of recombinant proteins R1, R2, and the B9 plastin and elastin blocks for yeast expression.
  • the generation of the B9 plastin and elastin blocks deviated from the described protocol in that a monomer library was initially generated based on a wobble base design and homologous sequences were obtained from SigmaGenosys. Seven recombinant genes were identified and 0.4 ug of each were used in multimerization reactions affording multimers ranging in size from 500-3000 bp with random incorporation of the monomers.
  • the pPICZ ⁇ A expression vector/XL100 Pichia expression strain will be utilized for yeast-B9 expression (see FIG. 29 ).
  • Recombinant plasmids containing R1 elastin and R2 plastin blocks will be isolated and digested with Bbs I/Xma I and Bsm B I/Xma I, respectively. The large fragment from each of these digestions will be isolate via preparative gel electrophoresis (1% agarose, 0.5 ⁇ TBE) and purified using the Zymoclean gel recovery kit. R1 and R2 fragments will be ligated by T4 DNA ligase, transformed into Top 10F′ and plated on LSLB plates under Zeocin resistance.
  • the Xma I site cuts within the Zeocin coding region, only clones containing the correctly assembled diblock (R2-R1), and thus, the correctly reassembled antibiotic coding region, will propagate.
  • the R2-R1 diblock is digested with Bsm B I/Xma I and the plasmid containing the R2 plastin block with Bbs I/Xma I. Similar protocols for ligation, transformation, and propagation will be followed. Via antibiotic selection, only colonies contain the correctly assembled triblock (R2-R1-R2) will survive (see FIG. 30 ).
  • the expression plasmid, pQE 80L (Qiagen, Inc) (see FIG. 17 ) will be prepared by deletion of the polyclonal region between the Bam H I and Hin d III restriction sites. A 75 bp adaptor containing flanking Lysine residues between which recombinant genes can be cloned will be inserted. The resulting plasmid will be defined as mpQE80L for the modification made to the original pQE 80L vector. This vector will be propagated in Top 10F′ and preparative amounts of DNA will be isolated.
  • Adaptor design In native elastin, crosslinking domains consist of poly-alanine and paired lysines.
  • This composition promotes an alpha helical structure and facilitates positioning for intermolecular crosslinking.
  • lysine residues can be incorporated for crosslinking.
  • Previous studies have indicated proper placement of lysine residues is essential for protein stabilization and adequate expression levels [26, 52, 78].
  • the pQE 80L expression vector will be utilized encoding an N-terminal oligonucleotide tag affording a strategy to incorporate lysines at the amino-terminus of the elastin genes.
  • Expression plasmids containing R1 and R2 will be used to transform the E coli expression strain DG99. Purification protocols will be adapted from those employed with BL21 (DE3) strain expressions utilizing elastin's inverse transition temperature, though the ability to purify using affinity chromatography is available. Large scale expression will be performed in an orbital shaker (225 rpm) at 37° C. in Terrific Broth medium supplemented with ampicillin (100 uL/mL) for 36-48 hours.
  • Cells will be harvested via centrifugation (4° C./8000 rpm/20 min) and the cell pellet resuspended in lysis buffer (64 mL; 100 mM NaCl, 50 mM Tris-HCl, pH 8.0) and stored at ⁇ 80° C. Frozen cells will be lysed via three freeze ( ⁇ 80° C.)/thaw cycles. Lysozme (1 mg/mL), benzonase (1 uL/10 mL), MgCl 2 (1 uL/mL), and protease inhibitor cocktail (1.3 mg/mL) will be added to the cell lysate and incubated at 37° C. for 30 minutes with constant agitation.
  • lysis buffer 64 mL; 100 mM NaCl, 50 mM Tris-HCl, pH 8.0
  • Frozen cells will be lysed via three freeze ( ⁇ 80° C.)/thaw cycles. Lysozme (1 mg/mL), benzonase
  • the lysed cells will be incubated at 4° C. for overnight followed by centrifugation (4° C./14000 rpm/20 min) for removal of cellular debris.
  • Repeatable purification protocols have been developed to purify elastin-like proteins by exploiting their solubility characteristics. In this way, proteins will be extracted from the cell lysate by three-five cycles of reversible temperature induced precipitation via centrifugation at 4° C./37° C. from 500 mM NaCl solution. Dialysis and lyophillization will follow with expected yields of 200-500 mg/L.
  • Gluteraldehyde crosslinking protocol has been adapted from the work of Welsh and Tirrell on elastin-like proteins [52]. Gluteraldehyde vapor phase crosslinking and solution phase crosslinking will be employed successively to crosslink elastin electrospun fabrics through the amine moieties of lysine residues. Fabrics will be enclosed in a chamber containing a pool of 12.5% gluteraldehyde (GTA) solution. Solution phase crosslinking will follow with submersion of the fabric in 10 mM GTA (in PBS, pH 7.4) for two hours at room temperature.
  • GTA gluteraldehyde
  • MALDI-TOF Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectroscopy
  • Turbidity measurements will be assessed as a function of temperature. Solutions of 0.5-0.7 mg/mL will be prepared from water and heated at rate of 1° C./min. The optical density will be measured at 280 nm by an Ultrospec 3000 UV/vis spectrophotometer equipped with a temperature controller (Amersham Pharmacia Biotech, Inc). The inverse transition temperature of the protein will be defined as the temperature associated with half-maximal turbidity [16].
  • a 5-18 weight % protein solution will be prepared by dissolving lyophilized protein in 2,2,2 trifluoroethanol (TFE) at room temperature.
  • the solution will be extruded at ambient temperature and pressure using a syringe pump (Havard Apparatus, Inc) at a flow rate of 150 ⁇ L/min though a positively charged needle (18G ⁇ 4 in).
  • a high voltage, low-current power supply (ES30P/DDPM, Gamma High Voltage Research, Inc) will be used to generate an electric potential gradient at approximately 18 kV.
  • the mandrel undergoes rotational and translational motion during the electrospinning process to create a nonwoven fabric conduit.
  • the electrospun conduit can be removed from the mandrel in the dehydrated state and used in subsequent experiments.
  • Four reference beads ( ⁇ 300 ⁇ m) will be attached to the surface of the ring, two on each wall. Following the placement of the beads, samples are loaded on two hooks in the ring testing apparatus, and strained to failure at a rate of 0.2 mm/sec. Using a step motor, strain is applied to the sample through downward displacement of the lower hook generating hook displacement data which is recorded through an analog/digital interface.
  • This data is used in conjunction with testing images captured by CCD camera to relate hook displacement to sample wall strain.
  • Force is recorded by a load transducer attached to the top hook and is normalized by initial cross-sectional area of the hydrated construct wall to calculate stress measurements.
  • Ultimate tensile strength is defined as the maximum stress withstood by samples with respect to the original cross-sectional area.
  • the elastic modulus is determined by the slope of the region extending between 25-75% of the ultimate tensile strength.
  • Fabric thickness will be measured in the hydrated state using an Advanced Rheometric Expansion System (ARES) (Rheometric Scientific) and verified by optical microscopy using the standard image analysis protocol.
  • Uniaxial Tension Six samples will be loaded by controlling displacement at a standard rate of 5 mm/min. As samples can be strained to only 70% engineering strain, only Young's Modulus can be obtained from this data set.
  • a miniature materials tester a Minimat 2000 (Rheometric Scientific) will be used in tensile deformation mode at a rate of 5 mm/min. Samples will be tested under ambient conditions and coated in a thin layer of mineral oil prior to loading to limit water loss during the test. Ultimate tensile strength and elastic modulus data can be obtained.
  • Hysteresis In these studies, three samples will be stretched to a predetermined strain, unloaded to a zero-stress state, and strained to 70% strain. Stress relaxation. Three samples will be stretched to a predetermined strain and held constant for times greater than one hour. The evolution of stress over time will be examined. Creep. Six samples will be subjected to a range of constant stresses for times approaching 24 hours. Material deformation over time will be assessed. Statistical Analysis. The Student's 2-tailed unpaired t-test will be utilized to evaluate data sets collected from different constructs to assess batch-to-batch variation. Additionally, Student's 2-tailed unpaired t-test will be used to establish statistical significance (p 0.05) of mechanical properties measured for crosslinked and non-crosslinked samples.
  • myeloperoxidase (MPO) (clone ab15484, Abcam) and Ham 56 (clone ab8186, Abcam) staining for neutrophils and macrophages, respectively, will be used to determine if an inflammatory response is generated.
  • Staining for endothelial factor VIII/von Willebrand factor (clone ab6994, Abcam) will be used to identify endothelial cells.
  • the surrounding tissue and the disc composition will be evaluated for inflammatory response, tissue ingrowth, and capsule formation. The observations will be ranked from 0 to 4, where 0 is a minimal and 4 is a maximal response. All observations will be made at a magnification of 200 ⁇ with five random areas observed per sample.
  • the proximal and distal IVC will be clamped and two rectangular segments measuring 15 ⁇ 33 mm of the anterior wall will be resected and replaced by an elastin patch and a control PTFE patch using a running suture technique with 5/0 Prolene.
  • the experimental elastin patch will be implanted superior to the control PTFE patch.
  • the control patch will be positioned superior to the elastin patch.
  • Three weeks post implantation the animals will be euthanized according to The American Veterinary Medical Association Guidelines.
  • the peritoneal cavity will be reentered though the previous wound and the implanted patches will be removed in an en-block manner [79-81].
  • the explanted patch will be sectioned according to requirements for mechanical testing as outlined in FIG. 31 . This will allow for two 4 ⁇ 13 mm samples to perform immunohistochemistry and microscopy studies. At the time of explantation, specimens will be photographed for measurements of thrombus free surface and overall pannus tissue ingrowth. Serial sections of the adjacent IVC and segments of the patch will be obtained for examination by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and light microscopy. Staining will be performed to examine endothelial and smooth muscle cell coverage, as well as associated cellular and matrix responses.
  • SEM Scanning Electron Microscopy
  • TEM Transmission Electron Microscopy
  • Staining will be performed to examine endothelial and smooth muscle cell coverage, as well as associated cellular and matrix responses.
  • immunohistochemical studies will include staining with endothelial factor VIII/von Willebrand (clone ab6994, Abcam) factor to identify endothelial cells, smooth muscle ⁇ -actin (clone ab9465, Abcam) to identify smooth muscle cells, and Ham 56 (clone ab8186, Abcam) to identify macrophages [82, 83].
  • Biomechanical testing will include tensile, mechanical hysteresis, and creep to be performed in triplicate. Data analysis will be conducted using ANOVA and Student's t-tests.
  • Sections measuring 3 ⁇ 3 mm will be sectioned from three sample areas within the graft. Samples will be fixed by immersion in 2.5% gluteraldehyde buffered with 0.1 M cacodylate (pH 7.5) for 4-16 hours and postfixed for one hour in 1% osmium tetroxide solution, washed, en block stained with uranyl acetate, dehydrated through graded concentrations of ethanol and embedded in embed 8-12 epoxy resin. Thin sections will be post stained with lead citrate and observed on a JEOL 1210 LaB 6 transmission electron microscope. The images will be captured on Kodak film and the negatives converted to digital images using an AGFA Duoscan T2500 scanner. TEM images will be processed using Adobe Photoshop [84].
  • Two 5 ⁇ 10 mm samples will be sectioned from the proximal and distal segments of the patch and surrounding vascular tissue. These samples will be fixed by immersion in oxygenated 2.5% glutaraldehyde buffered with 0.1 M cacodylate (pH 7.4) at 37° C. for 15 minutes followed by immersion overnight in glutaraldehyde. Tissue will be dehydrated through graded concentrations of ethanol and critical point dried. Samples will be inserted into an acetone filled specimen boat and transferred to a Polaron critical point drying apparatus where the exchange with liquid carbon dioxide was performed followed by decompression of CO 2 within the chamber.
  • Dried samples will be mounted on aluminum stubs and coated with a 10 nm thin film of gold palladium with a Denton DV-602 sputter coater.
  • Specimens will be imaged using the in-lens of a DS-130F field emission scanning electron microscope operated at 25 kV. SEM images of low (1000 ⁇ ) and intermediate (50,000 ⁇ ) magnifications will be digitally collected at a 17 Mb file size and Photoshop was used to adjust levels [84-86].
  • Elastin which is derived from the soluble precursor tropoelastin, is widely distributed in vertebrate tissues where it consists of repetitive glycine-rich hydrophobic elastomeric domains of variable length that alternate with alanine-rich, lysine-containing domains that form crosslinks [11-13].
  • Native elastin's intrinsic insolubility has restricted its capacity to be purified and processed into forms suitable for biomedical or industrial applications without extensive organic solvent and 2-mercatoethanol extractions, cyanogen bromide (CNBr) treatment, and enzymatic digestions. Recently, this limitation has been largely overcome, in part, by the structural characterization of the elastomeric domains.
  • VPGVG penta-
  • APGVGV hexapeptide
  • the protein sequences used to design these protein block copolymers were derived in part from a consideration of the primary structure of elastin. Specifically, we have synthesized and characterized a series of elastomeric triblock copolymers capable of virtual or physical crosslink formation. Proteins were synthesized that incorporate identical hydrophobic endblocks [VPAVG[(IPAVG) 4 (VPAVG)]] SEQ ID NO. 7, separated by a central hydrophilic block [(VPGVG) 2 (VPGEG)(VPGVG) 2 ] SEQ ID NO 52.
  • Elastin-coated grafts are characterized by contact angle goniometry, Fourier transform infrared (FT-IR) spectroscopy, and scanning electron microscopy (SEM) and their stability tested in a high shear rate environment.
  • FT-IR Fourier transform infrared
  • SEM scanning electron microscopy
  • the recombinant protein polymer B9 is derived from concatemerization of elastin-mimetic peptide sequences, expressed, and purified, as previously described [29, 34].
  • the structure consists of a triblock of form of [PN]-[X]-[PC], where
  • PN VPAVG[(IPAVG) 4 (VPAVG)] 16 IPAVG;
  • VPGVG VPGEG(VPGVG) 2 ] 48 VPGVG;
  • PC VPAVG[(IPAVG) 4 (VPAVG)] 16 IPAVG.
  • the elastin-mimetic polypeptide is run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie G250 stain (Bio-Rad). Molecular weight markers were Precision Plus Protein Kaleidoscope (Bio-Rad). As anticipated, the molecular weight of the recombinant triblock copolymer is ⁇ 180 kDa ( FIG. 18 ). Additional structural characterization data is been detailed elsewhere (29).
  • Elastin impregnation of an ePTFE vascular graft Impregnation of vascular grafts (4 mm i.d., Atrium Medical, Hudson, N.H.) is performed under positive pressure by clamping one end of the graft and infusing 5 mL of cold elastin polymer solution (6 w/v % in water) through the graft using a Luer-lok syringe. Elastin is extruded through the pores of the graft during this process. The prosthesis is subsequently immersed in an elastin bath at 4° C. for 6 hours to ensure uniform coating. After the 6 hour incubation, the graft is drained, 60 mL of air pushed through the lumen to remove excess elastin protein polymer, and the graft is oriented vertically at 37° C. for 30 min.
  • Multilayer coating of elastin films The elastin-impregnated ePTFE graft is post-coated by infusing 3 mL of chilled elastin solution (6 w/v % in water) through the open lumen of the graft. Using a Luer-lok syringe, 60 mL of air is pushed through the lumen to remove excess elastin, and the graft is then oriented vertically at 37° C. for 30 min. This process is repeated twice for a total of two post-coated layers. Samples are stored in warm saline.
  • graft samples are incubated for 10 min in Coomassie G250 stain (Bio-rad) in a 37° C. water bath and rinsed extensively with warm deionized water. Graft samples are sectioned lengthwise prior to staining.
  • Stability of protein polymer coating Stability of the prosthesis-bound protein film is evaluated in a closed-loop flow system by perfusing phosphate buffered saline (PBS) through the graft at 180 mL/min (500 s ⁇ 1 wall shear rate) at 37° C. for 24 hours.
  • PBS phosphate buffered saline
  • High resolution scanning electron micrographs Protein polymer coated ePTFE grafts are critical point dried, mounted onto aluminum specimen stubs with double-stick carbon tape, degassed for 30 minutes, and sputter coated with a 1 nm gold (Au) film. The film surface is examined using an in-lens field emission scanning electron microscope (ISI DS-130F Schottky Field Emission SEM) that was operated at 5 kV.
  • ISI DS-130F Schottky Field Emission SEM in-lens field emission scanning electron microscope
  • Infrared Spectroscopy Spectra are acquired using a Bio-Rad FTS-4000 Fourier Transform Infrared (FT-IR) spectrometer equipped with a wide band MCT detector, collected with 100 scans, and 2 cm ⁇ 1 resolution.
  • FT-IR Fourier Transform Infrared
  • Attenuated total reflectance (ATR) spectra of protein coated grafts were acquired using a Silvergate ATR anvil press accessory equipped with a germanium prism (Specac Inc., Woodstock, Ga.). The single beam spectrum of the ATR accessory is used as a background.
  • Spectra manipulations performed on the data such as baseline correction, CO 2 peak removal (from 2250-2405 cm ⁇ 1 ) and center-of-gravity frequency position determination of IR absorption bands were performed using the Grams/AI software package (Thermo Galactic Industries, Salem, N.H.).
  • Platelet Radiolabeling Autologous baboon platelets are radiolabeled on the day prior to the shunt study. Forty-five milliliters of whole blood is initially withdrawn into syringes containing 9 mL of acid citrate dextrose anticoagulant. The blood is centrifuged at 160 g for 15 min and the platelet rich plasma removed and centrifuged at 1500 g for 15 min. The platelet pellet is then removed, washed in normal saline solution with 0.1% (w/v) dextrose, and 600 ⁇ Ci of indium-111 oxine (Amersham Co.) is added to the platelet suspension.
  • Platelet Deposition Measurement Platelet uptake on test surfaces is monitored over a 60-min period using scintillation camera imaging of the 172 keV In g photon peak. A high-sensitivity 99 Tc collimator was utilized, and images are acquired with a GE 400T scintillation camera (General Electric, Milwaukee, Wis.) interfaced with a Medical Data Systems A3 image processing system (Ann Arbor, Mich.). Immediately before imaging, 5-min images are acquired of the 200 ⁇ L sample of platelet concentrate (injection standard) and of a segment of 4.0 mm i.d. Silastic tubing filled with autologous blood and having the same luminal volume as the test graft segment (blood standard). Images are obtained continuously with data storage at 5-min intervals. Deposited 111 In-platelet activity is calculated by subtracting the blood standard activity from all dynamic study images. Data are converted, at each time point, to total platelet deposition per unit test surface, as follows:
  • Platelets ⁇ / ⁇ unit ⁇ ⁇ surface ⁇ ⁇ area [ test ⁇ ⁇ surface ⁇ ⁇ area ⁇ ⁇ ( cpm ) - background ⁇ ⁇ activity ⁇ ⁇ ( cpm ) ] blood ⁇ ⁇ blood specific ⁇ ⁇ activity ⁇ ⁇ ( cpm ⁇ / ⁇ mL ) ⁇ ⁇ platelet ⁇ / ⁇ mL
  • Blood ⁇ ⁇ specific ⁇ ⁇ activity [ blood ⁇ ⁇ std ⁇ ⁇ ( cpm ) - backgournd ⁇ ⁇ ( cpm ) ] ⁇ ( 111 ⁇ ⁇ ln ⁇ ⁇ fraction ⁇ ⁇ in ⁇ ⁇ platelets ) vol ⁇ ⁇ of ⁇ ⁇ the ⁇ ⁇ blood ⁇ std ⁇ ⁇ ( mL )
  • Total fibrin accumulation was calculated by dividing the deposited 125 I-radioactivity (cpm) by the clottable fibrinogen radioactivity (cpm/mL) and multiplying by the circulating fibrinogen concentration (mg/mL) as measured in each experiment [36, 37].
  • FIG. 21 Infrared spectra of an uncoated ePTFE graft, a water-cast elastin film, an elastin-impregnated ePTFE graft, and an elastin-impregnated ePTFE graft post-coated with elastin are presented in FIG. 21 .
  • characteristic CF 2 antisymmetric and symmetric stretching modes at 1208 and 1147 cm ⁇ 1 , respectively are observed from the bare ePTFE graft ( FIG. 21A ).
  • Amide I and amide II stretching modes at 1646 and 1536 cm ⁇ 1 are typical of polypeptide films ( FIG. 21B ).
  • amide I and amide II stretching modes appear alongside CF 2 stretching modes ( FIGS. 21C and D).
  • Water contact angles were measured on the luminal surface on the graft. As anticipated, advancing/receding contact angles for the bare ePTFE graft were extremely high)(125/121° and decreased after elastin impregnation)(43/40° consistent with coverage of the ePTFE surface. These values agree with those measured for B9 elastin films cast from cold water)(47/42°. In contrast, Defife et al. obtained contact angles of 69° for surface grafted-(GVGVP) 100 on silicone rubber [8]. Contact angles of the post-coated graft could not be measured due to the hydrophilic nature of the coating with complete wetting of the film surface.
  • FIG. 22 Microstructural characterization of the elastin protein polymer film and elastin-mimetic impregnated vascular grafts using scanning electron microscopy Scanning electron micrographs (SEM) of impregnated and post-coated elastin grafts are presented in FIG. 22 .
  • SEM scanning electron microscopy Scanning electron micrographs
  • Total adsorbed fibrinogen during the test period was 0.03 ⁇ 0.02 mg/cm 2 for the elastin-coated grafts compared to 1.44 ⁇ 0.75 mg/cm 2 adsorbed on uncoated ePTFE grafts (p ⁇ 0.05).
  • triblock elastin-mimetic protein polymers is processable into multiple forms, including stable films that can be physically impregnated into ePTFE vascular prostheses generating a smooth luminal surface.
  • the capacity to incorporate amphiphilic drugs into these protein-based materials may provide an additional mechanism for the control of biological responses at blood- and tissue-materials [29].
  • a recombinant elastin-mimetic copolymer can be used to generate a hydrogel coating on the luminal surface of an ePTFE vascular prosthesis.
  • Elastin-based protein polymers are a promising class of materials characterized by high degree of biocompatibility, a tunable range of mechanical properties from plastic to elastic, a variety of processing options including gels, films, and nanofibers, and the potential for the incorporation of bioactive compounds within the polymer backbone itself or impregnated within a hydrogel.
  • elastin-mimetic materials will find utility in a number of vascular and non-vascular biomaterial applications.
  • TABLE 16 A summary of sequence listings is provided in TABLE 16. In an embodiment, the invention is directed to any one or more of these sequences.
  • TABLES 12-15 provide the various amino acid sequences and a corresponding DNA sequence for B10, lysB10, B9, and R4.
  • isotopic variants of compounds disclosed herein are intended to be encompassed by the disclosure.
  • any one or more hydrogens in a molecule disclosed can be replaced with deuterium or tritium.
  • Isotopic variants of a molecule are generally useful as standards in assays for the molecule and in chemical and biological research related to the molecule or its use. Methods for making such isotopic variants are known in the art. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently.
  • ionizable groups groups from which a proton can be removed (e.g., —COOH) or added (e.g., amines) or which can be quaternized (e.g., amines)]. All possible ionic forms of such molecules and salts thereof are intended to be included individually in the disclosure herein.
  • salts of the compounds herein one of ordinary skill in the art can select from among a wide variety of available counterions those that are appropriate for preparation of salts of this invention for a given application. In specific applications, the selection of a given anion or cation for preparation of a salt may result in increased or decreased solubility of that salt.
  • nucleotide sequences are specifically exemplified as DNA sequences, those sequences as known in the art are also optionally RNA sequences (e.g., with the T base replaced by U, for example).

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